Volume 6, Number 1—February 2000
Carbapenem-Hydrolyzing Metallo-ß-Lactamase from a Nosocomial Isolate of Pseudomonas aeruginosa in France
To the Editor: The carbapenems (meropenem and imipenem), the ß-lactams with the broadest spectrum, are stable to most ß-lactamases (1). Therefore, they are often used as antibiotics of last resort for treating nosocomial infections due to gram-negative bacteria resistant to other ß-lactams. Resistance to carbapenems and susceptibility to other ß-lactams in Pseudomonas aeruginosa is common as a result of reduced drug accumulation or increased expression of pump efflux (1).
Several extended-spectrum ß-lactamases have been reported in P. aeruginosa, but only two, IMP-1 and VIM-1, possess an extended hydrolysis profile that includes carbapenems (2-5). The chromosome-borne and plasmid-mediated carbapenem-hydrolyzing ß-lactamase, IMP-1, has been described in several gram-negative rods, including P. aeruginosa, P. cepacia, Alcaligenes xylosoxydans, and Enterobacteriaceae isolates in Japan (4,6). Recently, a chromosome-borne carbapenem-hydrolyzing ß-lactamase, VIM-1, was reported from a clinical isolate of P. aeruginosa in Italy (5), and uncharacterized carbapenem-hydrolyzing ß-lactamases have been reported in the United Kingdom and Portugal (7,8). The weakly related IMP-1 and VIM-1 (31.4% amino acid identity) are both zinc-dependent (metallo-enzymes) and confer resistance to all ß-lactams except monobactams (3,5).
In 1996, a 39-year-old French woman was hospitalized in Marseille for chronic myelogenous leukemia, pancytopenia, and allogeneic bone marrow transplantation. After a 15-day stay in the transplantation unit, fever developed and imipenem and amikacin were administered. Despite this treatment, the patient died of septic shock syndrome 5 days later. Three-day-old blood cultures grew a carbapenem-resistant P. aeruginosa isolate. This P. aeruginosa COL-1 isolate was resistant to most ß-lactams, including piperacillin/tazobactam, imipenem, meropenem, ceftazidime, cefepime (minimum inhibitory concentrations [MICs] of 128, 32, 16, 64, 32 mg/L, respectively), amikacin, tobramycin, gentamicin, netilmicin, and ciprofloxacin; however, the isolate was susceptible to aztreonam (MIC determination, genetic techniques and ß-lactamase assays are described elsewhere ). A sonicate of crude extract of P. aeruginosa COL-1 culture showed strong imipenem and meropenem hydrolysis activity (0.7 mU/mg and 1.9 mU/mg; reference P. aeruginosa strain <0.05 mU/mg) by UV spectrophotometry with 0.1 mM of substrate, after incubation in 50 mM phosphate buffer at 30°C. This activity was lost when the enzyme extract was preincubated with 10 mM of edetic acid and was partially restored by addition of 1 mM ZnCl2, indicating the presence of a metallo-carbapenem hydrolyzing ß-lactamase. Isoelectric focusing revealed two ß-lactamase bands of pI 5.6 and 9. Only the pI 5.6 ß-lactamase band was inhibited if the gel was overlaid with edetic acid before nitrocefin was added as the indicator substrate; the other pI 9 ß-lactamase likely corresponded to a naturally occurring AmpC cephalosporinase. This pI 5.6 value differed from the pI values of the carbapenem-hydrolyzing ß-lactamase previously reported in P. aeruginosa (3-5,7,8). Polymerase chain reaction amplification experiments were negative when internal primers were used for the only sequenced carbapenem-hydrolyzing ß-lactamase genes from P. aeruginosa encoding IMP-1 and VIM-1 and genomic DNA of P. aeruginosa COL-1. Transfer of the carbapenem resistance marker by conjugation to laboratory strains of P. aeruginosa or Escherichia coli was unsuccessful (9), but transformation by electroporation of a putative plasmid extract from P. aeruginosa COL-1 in E. coli, followed by selection onto amoxicillin-containing agar plates (9), gave a ca. 45-kb plasmid that produced the carbapenem-hydrolyzing ß-lactamase with a pI value of 5.6. Thus, the carbapenem-hydrolyzing ß-lactamase gene was plasmid-borne.
This case indicates the presence of a novel carbapenem-hydrolyzing ß-lactamase in P. aeruginosa in Europe, the first in France; its spread in gram-negative rods, as reported for IMP-1 in Japan, is of concern because, as seen in this case, routine laboratory detection is difficult and therapeutic options are extremely limited.
This work was supported by a grant from the Ministère de l'Education Nationale, de la Recherche et de la Technologie, Université Paris XI, Faculté de Médecine Paris Sud, UPRES-JE-2227, France.
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